Amino acid sequences capable of facilitating penetration across a biological barrier

ABSTRACT

This invention relates to novel pharmaceutical penetration compositions capable of facilitating penetration of at least one effector across biological barriers. The invention also relates to methods of treating or preventing diseases by administering penetration compositions to affected subjects.

RELATED APPLICATIONS

This application is a divisional application of U.S. Ser. No.10/665,184, filed Sep. 17, 2003, which is a continuation-in-part ofPCT/IB03/00968, filed on Feb. 7, 2003, which claims priority to U.S.Ser. No. 60/355,396, filed Feb. 7, 2002, each of which is incorporatedherein by reference in its entirety.

TECHNICAL FIELD OF THE INVENTION

This invention relates to novel penetration compositions capable offacilitating penetration of an effector across biological barriers.

BACKGROUND OF THE INVENTION

Techniques enabling efficient transfer of a substance of interest acrossa biological barrier are of considerable interest in the field ofbiotechnology. For example, such techniques may be used for thetransport of a variety of different substances across a biologicalbarrier regulated by tight junctions (i.e., the mucosal epithelia, whichincludes the intestinal and respiratory epithelia and the vascularendothelia, which includes the blood-brain barrier).

The intestinal epithelium represents the major barrier to absorption oforally administered compounds, e.g., drugs and peptides, into thesystemic circulation. This barrier is composed of a single layer ofcolumnar epithelial cells (primarily enterocytes, goblet cells,endocrine cells, and paneth cells), which are joined at their apicalsurfaces by the tight junctions. See Madara et al., PHYSIOLOGY OF THEGASTROINTESTINAL TRACT; 2^(nd) Ed., Johnson, ed., Raven Press, New York,pp. 1251-66 (1987).

Compounds that are presented in the intestinal lumen can enter the bloodstream through active or facilitative transport, passive transcellulartransport, or passive paracellular transport. Active or facilitativetransport occurs via cellular carriers, and is limited to transport oflow molecular weight degradation products of complex molecules such asproteins and sugars, e.g., amino acids, pentoses, and hexoses. Passivetranscellular transport requires partitioning of the molecule throughboth the apical and basolateral membranes. This process is limited torelatively small hydrophobic compounds. See Jackson, PHYSIOLOGY OF THEGASTROINTESTINAL TRACT; 2^(nd) Ed., Johnson, ed., Raven Press, New York,pp. 1597-1621 (1987). Consequently, with the exception of thosemolecules that are transported by active or facilitative mechanisms,absorption of larger, more hydrophilic molecules is, for the most part,limited to the paracellular pathway. However, the entry of moleculesthrough the paracellular pathway is primarily restricted by the presenceof the tight junctions. See Gumbiner, Am. J. Physiol., 253:C749-C758(1987); Madara, J. Clin. Invest., 83:1089-94 (1989).

Therefore, large hydrophilic molecules of therapeutic value present adifficult problem in the field of drug delivery. While they are readilysoluble in water, and thus easily dissolve in physiological media, suchmolecules are barred from absorption by the mucosal layer due to theircell-membrane impermeability. The epithelial cell membrane is composedof a phospholipid bilayer in which proteins are embedded via hydrophobicsegments. Thus, the cell membrane constitutes a very strong barrier fortransport of hydrophilic substances, including peptides and proteins.

A need remains for an efficient, specific, non-invasive, low-risk meansfor the delivery of biologically active molecules, such as polypeptides,drugs and other therapeutic agents, across various biological barriers.

SUMMARY OF THE INVENTION

The present invention provides penetration compositions containingtherapeutically active cationic or anionic impermeable molecules, inorder to enable their translocation across a biological barrier. Theinvention also relates to methods of using penetrating peptides totranslocate at least one effector across a biological barrier.

Specifically, the invention involves penetration compositions having atherapeutically effective amount of an effector, a counter ion to theeffector, and a penetrating peptide. Penetrating peptides have beendescribed in WO 03/066859, (PCT/IB03/00968), filed on Feb. 7, 2003, andin U.S. Ser. No. 60/355,396, filed Feb. 7, 2002, which are incorporatedherein by reference.

As used herein, a “penetration composition” includes any pharmaceuticalcomposition that facilitates the translocation of a substance, e.g., atleast one effector, across a biological barrier utilizing at least onecounter ion (i.e., an anionic counter ion or a cationic counter ion) anda penetrating peptide, as described herein. Examples of biologicalbarriers include, but are not limited to, tight junctions and the cellmembrane. Moreover, those skilled in the art will recognize thattranslocation may occur across a biological barrier in a tissue such asepithelial cells or endothelial cells.

The invention provides penetration compositions having apharmaceutically acceptable carrier or excipient, or a combinationthereof. In various embodiments, the penetration compositions of theinvention can be contained within a capsule, or can take the form of atablet, an aqueous dispersion, suspension, or emulsion, a cream, anointment, or a suppository. Likewise, the penetration composition can bedissolved in an at least partially water soluble solvent, such as, forexample, alcohols, (e.g., n-butanol, isoamyl (=isopentyl) alchohol,iso-butanol, iso-propanol, propanol, ethanol, ter-butanol), polyols,dmf, dmso, ethers, amides, esters, or various mixtures thereof.

Penetration compositions can include a penetrating peptide coupled to atleast one effector and can also include a suitable counter ion. The atleast one effector can be a therapeutically active cationic or anionicimpermeable molecule including, but not limited to, nucleic acids,glycosaminoglycans, proteins, peptides, or pharmaceutically activeagents, such as, for example, hormones, growth factors, neurotrophicfactors, anticoagulants, bioactive molecules, toxins, antibiotics,anti-fungal agents, antipathogenic agents, antigens, antibodies,antibody fragments, immunomodulators, vitamins, antineoplastic agents,enzymes, or therapeutic agents. For example, glycosaminoglycans actingas anionic impermeable compounds include, but are not limited to,heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, andhyaluronic acid. Nucleic acids serving as anionic impermeable moleculesinclude, but are not limited to, specific DNA sequences (e.g., codinggenes), specific RNA sequences (e.g., RNA aptamers, antisense RNA or aspecific inhibitory RNA (RNAi)), poly CpG, or Poly I:C syntheticpolymers of nucleic acids. Other suitable proteins include, but are notlimited to, hormones, gonadotropins, growth factors, cytokines,neurotrophic factors, immunomodulators, enzymes, anticoagulants, toxins,antigens, antipathogenic agents, antineoplastic agents, antibodies,antibody fragments, and other therapeutic agents. Specifically theseinclude, but are not limited to, insulin, erythropoietin (EPO),glucagon-like peptide 1 (GLP-1), αMSH, parathyroid hormone (PTH), growthhormone, calcitonin, interleukin-2 (IL-2), α1-antitrypsin,granulocyte/monocyte colony stimulating factor (GM-CSF), granulocytecolony stimulating factor (G-CSF), T20, anti-TNF antibodies, interferonα, interferon γ, lutenizing hormone (LH), follicle-stimulating hormone(FSH), enkephalin, dalargin, kyotorphin, basic fibroblast growth factor(bFGF), hirudin, hirulog, lutenizing hormone releasing hormone (LHRH)analog, brain-derived natriuretic peptide (BNP), and neurotrophicfactors.

As used herein, “cationic or anionic impermeable molecules” aremolecules that are positively (cationic) or negatively (anionic) chargedand are unable to efficiently cross biological barriers, such as thecell membrane or tight junctions. Preferably, cationic and anionicimpermeable molecules of the invention are of a molecular weight above200 Daltons. Anionic impermeable molecules are preferablyglycosaminoglycans, nucleic acids, or net negatively charged proteins,whereas cationic impermeable molecules are preferably net positivelycharged proteins. A protein's net charge is determined by twofactors: 1) the total count of acidic amino acids vs. basic amino acids,and 2) the specific solvent ph surroundings, which expose positive ornegative residues. As used herein, “net positively or net negativelycharged proteins” are proteins that, under non-denaturing pHsurroundings, have a net positive or net negative electric charge. Forexample, interferon β is a protein that contains 23 positively chargedresidues (lysines and arginines), and 18 negatively charged residues(glutamic or aspartic acid residues). Therefore, under neutral or acidicpH surroundings, interferon β constitutes a net positively chargedprotein. Conversely, insulin is a 51 amino acid protein that containstwo positively charged residues, one lysine and one arginine, and fourglutamic acid residues. Therefore, under neutral or basic pHsurroundings, insulin constitutes a net negatively charged protein. Ingeneral, those skilled in the art will recognize that all proteins maybe considered “net negatively charged proteins”, regardless of theiramino acid composition, depending on their ph and/or solventsurroundings. for example, different solvents can expose negative orpositive side chains depending on the solvent ph.

Penetration compositions according to the invention can also be used toenhance the penetration of smaller molecules that are otherwiseimpermeable through epithelial barriers. Examples of such moleculesinclude nucleic acids (i.e., DNA, RNA, or mimetics thereof), where thecounter ion is cationic. Conversely, when the counter ion is anionic,molecules such as Caspofungin, vitamin B12, and aminoglycosideantibiotics (e.g. Gentamycin, Amikacin, Tobramycin, or Neomycin) canpenetrate through epithelial barriers.

Counter ions of this invention can include, for example, anionic orcationic amphipathic molecules. In one embodiment, anionic or cationiccounter ions of this invention are ions that are negatively (anionic) orpositively (cationic) charged and can include a hydrophobic moiety.Under appropriate conditions, anionic or cationic counter ions canestablish electrostatic interactions with cationic or anionicimpermeable molecules, respectively. The formation of such a complex cancause charge neutralization, thereby creating a new uncharged entity,with further hydrophobic properties due to the inherent hydrophobicityof the counter ion.

Contemplated cationic counter ions include quaternary amine derivatives,such as benzalkonium derivatives. Suitable quaternary amines can besubstituted by hydrophobic residues. In general, quaternary aminescontemplated by the invention have the structure: 1-R1-2-R2-3-R3-4-R4-N,wherein R1, 2, 3, or 4 are alkyl or aryl derivatives. Further,quaternary amines can be ionic liquid forming cations, such asimidazolium derivatives, pyridinium derivatives, phosphonium compoundsor tetralkylammonium compounds. For example, imidazolium derivativeshave the general structure of 1-R1-3-R2-imidazolium where R1 and R2 canbe linear or branched alkyls with 1 to 12 carbons. Such imidazoliumderivatives can be further substituted for example by halogens or analkyl group. Specific imidazolium derivatives include, but are notlimited to, 1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium,1-hexyl-3-methylimidazolium, 1-methyl-3-octylimidazolium,1-methyl-3-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoroctyl)-imidazolium,1,3-dimethylimidazolium, and 1,2-dimethyl-3-propylimidazolium.

Pyridinium derivatives have the general structure of1-R1-3-R2-pyridinium where R1 is a linear or branched alkyl with 1 to 12carbons, and R2 is H or a linear or branched alkyl with 1 to 12 carbons.Such pyridinium derivatives can be further substituted for example byhalogens or an alkyl group. Pyridinium derivatives include, but are notlimited to, 3-methyl-1-propylpyridinium, 1-butyl-3-methylpyridinium, and1-butyl-4-methylpyridinium.

Suitable anionic counter ions are ions with negatively charged residuesderived from strong acids such as sulfonate or phosphonate, and furthercontain a hydrophobic moiety. Examples of such anionic counter ionsinclude sodium dodecyl sulphate or dioctyl sulfosuccinate.

The penetrating peptides used in penetration compositions of theinvention can have at least one amino acid sequence selected from:(BX)₄Z(BX)₂ZXB (SEQ ID NO:44); ZBXB₂XBXB₂XBX₃BXB₂X₂B₂ (SEQ ID NO:45);ZBZX₂B₄XB₃ZXB₄Z₂B₂ SEQ ID NO:46); ZB₉XBX₂B₂ZBXZBX₂ (SEQ ID NO:47);BZB₈XB₉X₂ZXB (SEQ ID NO:48); B₂ZXZB₅XB₂XB₂X₂BZXB₂ (SEQ ID NO:49);XB₉XBXB₆X₃B (SEQ ID NO:50); X₂B₃XB₄ZBXB₄XB_(n)XB (SEQ ID NO:51);XB₂XZBXZB₂ZXBX₃BZXBX₃B (SEQ ID NO:52); BZXBXZX₂B₄XBX₂B₂XB₄X₂ (SEQ IDNO:53); BZXBXZX₂B₄XBX₂B₂XB₄ (SEQ ID NO:54); B₂XZ₂XB₄XBX₂B₅X₂B₂ (SEQ IDNO:55); B_(q)X_(t)ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂ (SEQ ID NO:56);B₂ZX₃ZB_(m)X_(q)B₄XBX_(n)B_(m)ZB₂X₂B₂ (SEQ ID NO:57); X₃ZB₆XBX₃BZB₂X₂B₂(SEQ ID NO:58); and at least 12 contiguous amino acids of any of theseamino acid sequences, where X is any amino acid; B is a hydrophobicamino acid; and Z is a charged amino acid; and where q is 0 or 1; m is 1or 2; and n is 2 or 3; and where t is 1 or 2 or 3; and where thepenetrating peptide is capable of translocating across a biologicalbarrier.

Specifically, the penetrating peptide can have an amino acid sequence ofany one of SEQ ID NOS: 1-15 and 24-29. In another embodiment, theinvention provides a penetrating peptide having an amino acid sequenceof any one of SEQ ID NOS: 22, and 30-37. In addition, the penetratingpeptides of the invention include peptides having at least 12 contiguousamino acids of any of the peptides defined by SEQ ID NOS:1-15, 22, and24-37. The penetrating peptides can be less than thirty (30), less thantwenty-five (25), or less than twenty (20) amino acids in length. Theinvention also includes mutant or variant peptides any of whose residuesmay be changed from the corresponding residues shown in SEQ ID NOS:1-15, 22, and 24-37, while still encoding a peptide that maintains itspenetrating activities and physiological functions, or functionalfragments thereof. For example, the fragment of an amino acid sequenceof any one of SEQ ID NOS: 1-15, 22 and 24-37 is at least 10 amino acidsin length, and may contain conservative or non-conservative amino acidsubstitutions.

In general, a penetrating peptide variant that preserves thetranslocating function includes any variant in which residues at aparticular position in the sequence have been substituted by other aminoacids, and further includes the possibility of inserting an additionalresidue or residues between two residues of the parent protein as wellas the possibility of deleting one or more residues from the parentsequence. Any such amino acid substitution, insertion, or deletion isencompassed by the invention. In favorable circumstances, thesubstitution is a conservative substitution.

Amino acid substitutions at “non-essential” amino acid residues can bemade in the penetrating peptides. A “non-essential” amino acid residueis a residue that can be altered from the native sequences of thepenetrating peptides without altering their biological activity, whereasan “essential” amino acid residue is required for such biologicalactivity. For example, amino acid residues that are conserved among thepenetrating peptides of the invention are predicted to be particularlynon-amenable to substantial alteration. Amino acids for whichconservative substitutions can be made are well known within the art.

Mutations can be introduced into nucleic acids encoding penetratingpeptides by standard techniques, including, but not limited tosite-directed mutagenesis and PCR-mediated mutagenesis. Preferably,conservative amino acid substitutions are made at one or more predicted,non-essential amino acid residues. A “conservative amino acidsubstitution” is one in which the amino acid residue is replaced with anamino acid residue having a similar side chain. Families of amino acidresidues having similar side chains have been defined within the art.These families include amino acids with basic side chains (e.g., lysine,arginine, histidine), acidic side chains (e.g., aspartic acid, glutamicacid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine). Thus, a predicted non-essentialamino acid residue in the penetrating peptide is replaced with anotheramino acid residue from the same side chain family.

Alternatively, mutations can be introduced randomly along all or part ofa penetrating peptide coding sequence, such as by saturationmutagenesis, and the resultant mutants can be screened for biologicalactivity to identify mutants that retain activity. Followingmutagenesis, the encoded penetrating peptide can be expressed by anyrecombinant technology known in the art and the activity of the proteincan be determined. Amino acid substitutions can also be introducedduring artificial peptide synthesis such as solid-phase synthesis ofpeptides.

The relatedness of amino acid families may also be determined based onside chain interactions. Substituted amino acids may be fully conserved“strong” residues or fully conserved “weak” residues. The “strong” groupof conserved amino acid residues may be any one of the following groups:STA, NREQK (SEQ ID NO:17), NHQK (SEQ ID NO:18), NDEQ (SEQ ID NO:19),QHRK (SEQ ID NO:20), MILV (SEQ ID NO:21), MILF (SEQ ID NO:23), HY, FYW,wherein the single letter amino acid codes are grouped by those aminoacids that may be substituted for each other. Likewise, the “weak” groupof conserved residues may be any one of the following: CSA, ATV, SAG,STNK (SEQ ID NO:38), STPA (SEQ ID NO:39), SGND (SEQ ID NO:40), SNDEQK(SEQ ID NO:41), NDEQHK (SEQ ID NO:42), NEQHRK (SEQ ID NO:43), HFY,wherein the letters within each group represent the single letter aminoacid code.

The penetrating peptides utilized herein are preferably modified byhydrophobic moieties. A hydrophobic agent can be a single molecule or acombination of hydrophobic molecules, like aliphatic or aromaticmolecules. Examples of aliphatic hydrophobic agents include fatty acids,mono-, di-, or tri-glycerides, ethers, or cholesterol esters of fattyacids. The tri-glyceride can be tricaprin, for example. An example of anaromatic hydrophobic agent includes benzyl benzoate. The penetratingpeptides are then incorporated into the construct of the penetrationcomposition, including the desired effector. The hydrophobization of thepenetrating peptide can be achieved via acylation of free amino group(s)of extra lysine(s), interspaced by glycine, alanine, or serine residues,added at the C-terminus of the penetrating peptide. Acylation of thepenetrating peptide preferably utilizes long-chain fatty acids such asstearoyl, palmitoyl, oleyl, ricinoleyl, or myristoyl.

The penetrating peptides of the invention can also include amino acidanalogs in which one or more peptide bonds have been replaced with analternative type of covalent bond (a “peptide mimetic”) that is notsusceptible to cleavage by peptidases elaborated by the subject. Whereproteolytic degradation of a peptide composition is encounteredfollowing administration to the subject, replacement of one or moreparticularly sensitive peptide bonds with a noncleavable peptide mimeticrenders the resulting peptide derivative compound more stable, and thus,more useful as a therapeutic. Such mimetics, and methods ofincorporating them into peptides, are well known in the art.

Similarly, the replacement of an L-amino acid residue by a D-amino acidresidue is one standard method for rendering the compound less sensitiveto enzymatic destruction. Other amino acid analogs are known in the art,such as norleucine, norvaline, homocysteine, homoserine, ethionine, andthe like. Also useful is derivatizing the compound with anamino-terminal blocking group such as a t-butyloxycarbonyl, acetyl,methyl, succinyl, methoxysuccinyl, suberyl, adipyl, azelayl, dansyl,benzyloxycarbonyl, fluorenylmethoxycarbonyl, methoxyaselayl,methoxyadipyl, methoxysuberyl, and a 2,3-dinitrophenyl group.

The penetrating peptides of the invention can also be further chemicallymodified. For example, one or more polyethylene glycol (PEG) residuescan be attached to the penetrating peptides of the invention.

The penetration composition involves the coupling of the penetratingpeptide to the effector, directly or indirectly. As used herein, theterm “coupled” is meant to include all such specific interactions thatresult in two or more molecules showing a preference for one anotherrelative to some third molecule, including any type of interactionenabling a physical association between an effector and a penetratingpeptide. Preferably this includes, but is not limited to, electrostaticinteractions, hydrophobic interactions and hydrogen bonding, but doesnot include non-specific associations such as solvent preferences. Theassociation must be sufficiently strong so that the effector does notdissociate before or during penetration of the biological barrier.

Furthermore, the coupling of the effector to the penetrating peptide canbe achieved indirectly via a mediator. For example, such a mediator canbe a large hydrophobic molecule, such as a triglyceride, that binds theeffector-counter ion complex, on the one hand, and the hydrophobizedpenetrating peptide, on the other hand.

The invention also includes methods of producing a penetrationcomposition by coupling a therapeutically effective amount of at leastone effector with a penetrating peptide and a counter-ion to theeffector. Such coupling can be via a non-covalent bond. The non-covalentbond can be achieved by adding a hydrophobic moiety to the penetratingpeptide, such that the moiety enables the penetrating peptide to beincorporated at the interface of the hydrophobic vesicle in which theeffector is contained.

In one embodiment, the compositions of the invention can be prepared vialyophilization of the effector (supplied under preferred pHsurroundings) and the counter ion. The composition can be furthersupplemented by a polyanionic molecule, such as phytic acid, and/or anyother constituent of the pharmaceutical excipient or carrier, which canbe optionally added with the effector and counter ion during thelyophilization. The lyophilized materials can then be reconstitutedunder preferred solvent surroundings. During the reconstitution, otherconstituents, including one or more of the penetrating peptides, can beadded. Other constituents can include, for example, N-methyl pirolidine,cremophore, tricaprin, pluronic F-68, aprotinin, solutol HS-15, N-acetylCysteine, sodium hydroxide, acetic acid, sodium acetate and/orL-Arginine.

The invention also involves methods of translocating an effector acrossa biological barrier by using the penetration compositions of theinvention. For example, an effector can be coupled to penetrationcompositions according to the invention, which can then be introduced toa biological barrier, thereby effectively translocating the effectoracross the biological membrane.

As used herein, the term “biological barrier” is meant to includebiological membranes such as the plasma membrane as well as anybiological structures sealed by tight junctions (or occluding junctions)such as the mucosal epithelia, including, but not limited to, theintestinal or respiratory epithelia or the vascular endothelia,including, but not limited to, the blood-brain barrier.

The invention further includes a pharmaceutical composition containing atherapeutically or prophylactically effective amount of one or morepenetrating peptides, an effector, a suitable counter ion, andadditional pharmaceutically acceptable constituents. These additionalconstituents can assist either in the construction, solubility, ormaintenance of the penetration composition. The pharmaceuticalcomposition can further include a suitable carrier(s) and additives thatprotect the penetration composition such as protease inhibitors or aprotection against the digestive environment of the gastrointestinaltract, such as enteric coatings. Specifically, such additionalconstituents include, but are not limited to, a poloxamer, N-acetylcysteine (NAC), Aprotinin, and Solutol HS 15.

Preferred “pharmaceutical compositions” include, e.g., enteric-coatedtablets and gelatin capsules comprising the active ingredient togetherwith a) diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine; b) protease inhibitors such as Aprotinin ortrasylol; c) lubricants, e.g., silica, talcum, stearic acid, itsmagnesium or calcium salt, poloxamer and/or polyethyleneglycol; fortablets also d) binders, e.g., magnesium aluminum silicate, starchpaste, gelatin, tragacanth, methylcellulose, sodiumcarboxymethylcellulose and/or polyvinylpyrrolidone; e) ionic surfaceactive agents such as poloxamer, Solutol HS15, Cremophore, and bileacids, if desired f) disintegrants, e.g., starches, agar, alginic acidor its sodium salt, or effervescent mixtures; and/or g) absorbents,colorants, flavors and sweeteners. Suppositories are advantageouslyprepared from fatty emulsions or suspensions. The compositions may besterilized and/or contain adjuvants, such as preserving, reducing agentse.g., NAC (N-Acetyl-L-Cysteine), stabilizing, wetting or emulsifyingagents, solution promoters, salts for regulating the osmotic pressureand/or buffers. In addition, they may also contain other therapeuticallyvaluable substances. The compositions are prepared according toconventional mixing, granulating or coating methods, respectively, andcontain about 0.01 to 75%, preferably about 0.1 to 10%, of the activeingredient.

These compositions may further contain a mixture of at least twosubstances selected from the group consisting of a non-ionic detergent,an ionic detergent, a protease inhibitor, and a reducing agent. Forexample, the non-ionic detergent may be a poloxamer or Solutol HS 15;the poloxamer may be pluronic F-68; the ionic detergent may be a bilesalt; the bile salt may be Taurodeoxycholate; the protease inhibitor maybe selected from the group consisting of aprotonin and soy bean trypsininhibitor; and/or the reducing agent may be NAC.

Other suitable protease inhibitors that can be added to the penetrationcomposition are described in Bernkop-Schnurch et al., J. Control.Release, 52:1-16 (1998). These include, e.g., inhibitors of luminallysecreted proteases, examples of which are aprotinin, Bowman-Birkinhibitor, soybean trypsin inhibitor, chicken ovomucoid, chickenovoinhibitor, human pancreatic trypsin inhibitor, camostate mesilate,flavonoid inhibitors, antipain, leupeptin, p-aminobenzamidine, AEBSF,TLCK, APMSF, DFP, PMSF, poly(acrylate) derivatives, chymostatin,benzyloxycarbonyl-Pro-Phe-CHO, FK-448, sugar biphenylboronic acidscomplexes, β-phenylpropionate, elastatinal,methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK) (SEQ IDNO:66), EDTA, and chitosan-EDTA conjugates. These also includeinhibitors of membrane bound proteases, such as amino acids, di- andtripeptides, amastatin, bestatin, puromycin, bacitracin, phosphinic aciddipeptide analogues, α-aminoboronic acid derivatives, Na-glycocholate,1,10-phenantroline, acivicin, L-serine-borate, thiorphan, andphosphoramidon.

The invention also provides kits having one or more containerscontaining a therapeutically or prophylactically effective amount of apharmaceutical composition or of a penetration composition of theinvention.

Also described are methods of treating or preventing a disease orpathological condition by administering to a subject in which suchtreatment or prevention is desired, a penetration composition in anamount sufficient to treat or prevent the disease or pathologicalcondition. For example, the disease or condition to be treated mayinclude but are not limited to endocrine disorders, including diabetes,infertility, hormone deficiencies and osteoporosis; neurodegenerativedisorders, including Alzheimer's disease and other forms of dementia,Parkinson's disease, multiple sclerosis, and Huntington's disease;cardiovascular disorders, including atherosclerosis, hyper- andhypocoagulable states, coronary disease, and cerebrovascular events;metabolic disorders, including obesity and vitamin deficiencies; renaldisorders, including renal failure; haematological disorders, includinganemia of different entities; immunologic and rheumatologic disorders,including autoimmune diseases, and immune deficiencies; infectiousdiseases, including viral, bacterial, fungal and parasitic infections;neoplastic diseases; and multi-factorial disorders, including impotence,chronic pain, depression, different fibrosis states, and short stature.

Also provided are methods of oral or nasal, i.e., mucosal, vaccinationinvolving administering to a subject in need of vaccination an effectiveamount of a penetration composition of the invention, wherein theeffector includes an antigen to which vaccination is desired. In oneembodiment, the effector can be a protective antigen (PA) for use in avaccine against Anthrax. In another embodiment, the effector can be aHepatitis B surface antigen (HBs) for use in a vaccine against HepatitisB.

The invention also includes penetrating peptides that are derived from abacterial protein. In one embodiment, the invention provides apenetrating peptide derived from a bacterial protein having an aminoacid sequence of any one of SEQ ID NOS:1-8, 10-15 and 25-29. Such apenetrating peptide can be derived from an integral membrane protein, abacterial toxin, or an extracellular protein. The penetrating peptidecan also be derived from a human neurokinin receptor. In anotherembodiment, the invention provides a peptide derived from a neurokininreceptor having an amino acid sequence of any one of SEQ ID NOS:9 and24.

The details of one or more embodiments of the invention have been setforth in the accompanying description below. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methodsand materials are now described. Other features, objects, and advantagesof the invention will be apparent from the description and from theclaims. In the specification and the appended claims, the singular formsinclude plural referents unless the context clearly dictates otherwise.Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All patents and publicationscited in this specification are incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows amino acid sequence alignment of ORF HI0638 and itshomologues from other pathogenic bacteria.

FIG. 2 shows an amino acid sequence alignment of the penetratingpeptides used in this invention, as well as their organism of origin.

FIG. 3 shows a graph of blood glucose levels in mice plotted againsttime, following insulin translocation across epithelial cell membranesvia administration of penetration compositions of the invention.

FIG. 4 shows a graph of blood glucose levels in rats plotted againsttime, following insulin translocation across epithelial cell membranesvia administration of penetration compositions of the invention.

DETAILED DESCRIPTION OF THE INVENTION

The use of small peptide carriers such as the penetration compositionsdescribed herein allow for high quality and purity, low immunogenicityand the potential for highly efficient delivery through biologicalbarriers in an organism. Accordingly, peptide carriers have thepotential to improve upon conventional transporters such as liposomes orviruses for the efficient delivery of many macromolecules. The presentinvention employs a short peptide motif to create penetrationcompositions to specifically transport macromolecules across biologicalbarriers sealed by tight junctions.

The present invention provides a peptide penetration system, i.e., apenetration composition, that specifically targets various tissues,especially epithelial and endothelial ones, for the delivery of drugsand other therapeutic agents across a biological barrier. Existingtransport systems known in the art are too limited to be of generalapplication because they are inefficient, they alter the biologicalproperties of the active substance, they kill the target cell, theyirreversibly destroy the biological barrier and/or they pose too high ofa risk to be used in human subjects.

The peptide penetration system of the present invention uses conservedpeptide sequences from various proteins involved in paracytosis tocreate a penetration composition capable of crossing biologicalbarriers. For example, a peptide encoded by or derived from ORF HI0638of Haemophilus influenzae facilitates penetration of this bacteriumbetween human lung epithelial cells without compromising the epithelialbarrier. The peptide sequence encoded by ORF HI0638 is conserved incommon pathogenic bacteria or symbiotic bacteria including, for example,Haemophilus influenzae, Pasteurella multocida, Escherichia coli, Vibriocholerae, Buchnera aphidicola, Pseudomonas aeruginosa, and Xylellafastidiosa. A peptide homologous to the N-terminal sequence of HI0638 isalso found in other bacteria including, for example, Rhizobium loti,Chlamydia pneumoniae, NprB from Bacillus subtilis, and pilins fromKingella dentrificans and Eikenella corrodens.

Furthermore, a similar peptide sequence is also conserved in proteins ofeukaryotic origin such as the neurokinin receptor family proteins,including the human NK-1 and NK-2 receptors. It is known that theneurokinin receptor family is involved in the control of intercellularpermeability including plasma extravasation and oedema formation.Extravasation, the leakage and spread of blood or fluid from vesselsinto the surrounding tissues, often follows inflammatory processesinvolved in tissue injury, allergy, burns and inflammation. Inparticular, when NK-1 receptors on blood vessels are activated, skininflammation occurs due to an increase in vascular permeability. SeeInoue, et al., Inflamm. Res., 45:316-323 (1996). The neurokinin NK-1receptor also mediates dural and extracranial plasma proteinextravasation, thereby implicating the NK-1 receptor in thepathophysiology of migraine headache. See O'Shaughnessy and Connor,Euro. J. of Pharm., 236:319-321 (1993).

The sequences of example penetrating peptides of the invention are shownin Tables 1 and 2. TABLE 1 Peptide/Organism Sequence SEQ ID NO Peptide1: from ORF H10638 NYHDIVLALAGVCQSAKLVHQLA (SEQ ID NO:1) Haemophilusinfluenzae Peptide 2: from PM1850 NYYDITLALAGVCQAAKLVQQFA (SEQ ID NO:2)Pasteurella multocida Peptide 3: from YCFC NYYDITLALAGICQSARLVQQLA (SEQID NO:3) Escherichia coli Peptide 4: from VC1127 AIYDRTIAFAGICQAVALVQQVA(SEQ ID NO:4) Vibrio cholerae Peptide 5: from BU262KIHLITLSLAGICQSAHLVQQLA (SEQ ID NO:5) Buchnera aphidicola Peptide 6:from PA2627 DPRQQLIALGAVFESAALVDKLA (SEQ ID NO:6) Pseudomonas aeruginosaPeptide 7: from XF1439 LIDNRVLALAGVVQALQQVRQIA (SEQ ID NO:7) Xylellafastidiosa Peptide 8: from MLR0187 NLPPIVLAVIGICAAVFLLQQYV (SEQ ID NO:8)Rhizobium loti Peptide 9: from Human NK-2 NYFIVNLALADLCMAAFNAAFNF (SEQID NO:9) Receptor Peptide 10: from CPN0710/C TAFDFNKMLDGVCTYVKGVQQYL(SEQ ID NO:10) Chlamydia pneumoniae Peptide 11: from MLR4119RAILIPLALAGLCQVARAGDISS (SEQ ID NO:11) Rhizobium loti Peptide 12: fromNprB MRNLTKTSLLLAGLCTAAQMVFVTH (SEQ ID NO:12) Bacillus subtilis Peptide13: from Pilin IELMIVIAIIGILAAIALPAYQEYV (SEQ ID NO:13) Kingelladentrificans Peptide 14: from Pilin IELMIVIAIIGILAAIALPAYQDYV (SEQ IDNO:14) Eikenella corrodens Peptide 15: from zonula ASFGFCIGRLCVQDGF (SEQID NO:15) occludens toxin (ZOT) Peptide 29: from Human NK-1NYFLVNLAFAEASMAAFNTVVNF (SEQ ID NO:24) Receptor Peptide 30: from YCFCMNYYDITLALAGICQSARLVQQLA (SEQ ID NO:25) Escherichia coli Peptide 31:from YCFC MYYDITLALAGICQSARLVQQLA (SEQ ID NO:26) Escherichia coliPeptide 32: from YCFC MYDITLALAGICQSARLVQQLA (SEQ ID NO:27) Escherichiacoli Peptide 33: from NprB MRNLTRTSLLLAGLCTAAQMVFV (SEQ ID NO:28)Bacillus subtilis Peptide 34: from ORF H10638 NYHDIVLALAGVCQSARLVHQLA(SEQ ID NO:29) Haemophilus influenzae

The penetrating peptides of the instant invention also include peptidescontaining at least 12 contiguous amino acids of any of the peptidesdefined by SEQ ID NOS:1-15 and 24-29. TABLE 2 SEQ Peptide's ID name NO.Sequence IBW-002 22 AcNYYDITLALAGICQSARLVQQLAGGGKGGKNH₂ IBW-003 30AcNLPPIVLAVIGICAAVFLLQQYVGGGKGGKNH₂ IBW-004 31AcNYFIVNLALADLCMAAFNAAFNFGGGKGGKNH₂ IBW-005 32AcMRNLTRTSLLLAGLCTAAQMVFVGGGKGGKNH₂ IBW-006 33AcNYHDIVLALAGVCQSARLVHQLAGGKGGKNH₂ IBW-007 34AcNYFLVNLAFAEASMAAFNTVVNFGGKGGKNH₂ IBW-002V1 35AcMNYYDITLALAGICQSARLVQQLAGGGKGGKNH₂ IBW-002V2 36AcMYYDITLALAGICQSARLVQQLAGGGKGGKNH₂ IBW-002V3 37AcMYDITLALAGICQSARLVQQLAGGGKGGKNH₂

The penetration compositions of the present invention exhibit efficient,non-invasive delivery of an unaltered biologically active substance, andthus, have many uses. For example, the penetrating peptides of theinvention can be used in the treatment of bacterial infections. Sincethe introduction of the penicillins, pathogenic bacteria have beensteadily acquiring novel mechanisms enabling a growing resistance toantibiotic therapy. The expanding number of highly insensitive bacterialpathogens presents an ever-growing challenge to physicians andcaregivers. Consequently, patients are often forced to remainhospitalized for long periods, in order to receive IV antibiotictherapy, with obvious economic and medical disadvantages. Aminoglycosideantibiotics are potent antibacterial antibiotics, that are ineffectivelyabsorbed through biological barriers. The penetration compositions ofthe invention can be used to deliver aminoglycosides, such asgentamycin, tobramycin, neomycin, and amikacin, across the mucosalepithelia at high yield.

Furthermore, the penetrating peptides of the invention can be used inthe treatment of diabetes. Insulin levels in the blood stream must betightly regulated. The penetration compositions of the invention can beused to deliver insulin across the mucosal epithelia at high yield.Alternative non-invasive insulin delivery methods, previously known inthe art, have typical yields of 1-5% and cause intolerable fluctuationsin the amount of insulin absorbed. A more innovative treatment forelevated blood glucose levels involves the use of glucagon-likepeptide 1. GLP-1 is a potent hormone, which is endogenously secreted inthe gastrointestinal tract upon food injection. Its importantphysiological action is to augment the secretion of insulin in aglucose-dependant manner, thus encompassing a novel treatment fordiabetic states.

In addition, these penetration compositions also can be used to treatconditions resulting from atherosclerosis and the formation of thrombiand emboli such as myocardial infarction and cerebrovascular accidents.Specifically, the penetration compositions can be used to deliverheparin across the mucosal epithelia. Heparin is an established,effective and safe anticoagulant. However, its therapeutic use islimited by the need for parenteral administration. Thus far there hasbeen limited success in the direction of increasing heparin absorptionfrom the intestines, and a sustained systemic anticoagulant effect hasnot been achieved.

The penetration composition of this invention can also be used to treathematological diseases and deficiency states that are amenable byadministration of hematological growth factors. Erythropoietin is aglycoprotein which stimulates red blood cell production. It is producedin the kidney and stimulates the division and differentiation ofcommitted erythroid progenitors in the bone marrow. Endogenously,hypoxia and anemia generally increase the production of erythropoietin,which in turn stimulates erythropoiesis. However, in patients withchronic renal failure (CRF), production of erythropoietin is impaired,and this erythropoietin deficiency is the primary cause of their anemia.Recombinant EPO stimulates erythropoiesis in anemic patients with CRF,including patients on dialysis as well as those who do not requireregular dialysis. Additional anemia states treated by EPO includeZidovudine-treated HIV-infected patients, cancer patients onchemotherapy. Anemia in cancer patients may be related to the diseaseitself or the effect of concomitantly administered chemotherapeuticagents.

Another widespread cause of anemia is pernicious anemia, caused by alack of vitamin B12. The complex mechanism of vitamin B12 absorption inthe gastrointestinal tract involves the secretion and binding toIntrinsic Factor. This process is abnormal in pernicious anemiapatients, resulting in lack of vitamin B12 absorption and anemia. Thepenetration compositions of the invention can be used to deliver vitaminB12 across the mucosal epithelia at high yield.

Colony stimulating factors are glycoproteins which act on hematopoieticcells by binding to specific cell surface receptors and stimulatingproliferation, differentiation, commitment, and some end-cell functionalactivation. G-CSF regulates the production of neutrophils within thebone marrow and affects neutrophil progenitor proliferation,differentiation and selected end-cell functional activation, includingenhanced phagocytic ability, priming of the cellular metabolismassociated with respiratory burst, antibody dependent killing, and theincreased expression of some functions associated with cell surfaceantigens.

In cancer patients, recombinant granulocyte colony-stimulating factorhas been shown to be safe and effective in accelerating the recovery ofneutrophil counts following a variety of chemotherapy regimens, thuspreventing hazardous infectious. G-CSF can also shorten bone marrowrecovery when administered after bone marrow transplantations.

The penetration composition of this invention can also be used toadminister monoclonal antibodies for different indications. For example,administration of antibodies that block the signal of tumor necrosisfactor (TNF) can be used to treat pathologic inflammatory processes suchas rheumatoid arthritis (RA), polyarticular-course juvenile rheumatoidarthritis (JRA), and the resulting joint pathology.

Additionally, the penetration compositions of this invention can be usedto treat osteoporosis. It has recently been shown that intermittentexposure to parathyroid hormone (PTH), as occurs in recombinant PTHinjections, results in an anabolic response, rather than the well knowncatabolic reaction induced by sustained exposure to elevated PTH levels,as seen in hyperparathyroidism. Thus, non invasive administration of PTHmay be beneficial for increasing bone mass in various deficiency states,like osteoporosis. See Fox, Curr. Opin. Pharmacol., 2:338-344 (2002).

Currently, the delivery of effectors (e.g., the delivery of gentamycin,insulin, heparin, or erythropoietin to the blood stream) requiresinvasive techniques such as intravenous or intramuscular injections. Oneadvantage of the penetration composition is that it can delivereffectors across biological barriers through non-invasiveadministration, including, for example oral, bucal, rectal, inhalation,insufflation, transdermal, or depository. In addition, a furtheradvantage of the penetration composition of the invention is that it cancross the blood-brain barrier, thereby delivering effectors to thecentral nervous system (CNS).

The peptides described herein serve as the basis for the design oftherapeutic “cargos”, namely the coupling of the carriers (“penetratingpeptide”) with one or more therapeutic agents (“effectors”). Preferablya non-covalent bond is used to couple a penetrating peptide to one ormore effectors. The penetrating peptide can be attached to a linker towhich imaging compounds can be covalently attached, for example throughfree amino groups of lysine residues. Such a linker includes, but is notlimited to, the amino acid sequence GGKGGK (SEQ ID NO:16), alternativelyreferred to herein as IBW-001).

A penetration composition is a composition that facilitates the passage,translocation, or penetration of a substance across a biologicalbarrier, particularly through or between cells “sealed” by tightjunctions. Translocation may be detected by any method known to thoseskilled in the art, including using imaging compounds, such asradioactive tagging, and/or fluorescent probes or dyes, incorporatedinto a penetration composition in conjunction with a paracytosis assayas described in, for example, Schilfgaarde, et al., Infect. and Immun.,68(8):4616-23 (2000). Generally, a paracytosis assay is performed by: a)incubating a cell layer with a penetration composition; b) making crosssections of the cell layers; and c) detecting the presence of thepeptides or penetration compositions. The detection step may be carriedout by incubating the fixed cell sections with labeled antibodiesdirected to the peptide, followed by detection of an immunologicalreaction between the peptide and the labeled antibody. Alternatively,the peptide may be labeled using a radioactive label, or a fluorescentlabel, or a dye in order to directly detect the presence of the peptide.Further, a bioassay can be used to monitor the peptide translocation.For example, using a bioactive molecules such as erythropoietin,included in a penetration composition, the increase in hemoglobin orhematocrit can be measured. Similarly, by using a bioactive moleculesuch as insulin coupled with a penetration composition, the drop inblood glucose level can be measured.

As used herein, the term “effector” refers to any cationic or anionicimpermeable molecule or compound of, for example, biological,therapeutic, pharmaceutical, or diagnostic tracing. An anionicimpermeable molecule can consist of nucleic acids (ribonucleic acid,deoxyribonucleic acid) from various origins, and particularly of human,viral, animal, eukaryotic or prokaryotic, plant, synthetic origin, etc.A nucleic acid of interest may be of a variety of sizes, ranging from,for example, a simple trace nucleotide to a genome fragment, or anentire genome. It may be a viral genome or a plasmid.

Alternatively, the effector of interest can be a protein, such as, forexample, an enzyme, a hormone, a cytokine, an apolipoprotein, a growthfactor, a bioactive molecule, an antigen, or an antibody, etc. As usedherein, the term “bioactive molecule” refers to those compounds thathave an effect on or elicit a response from living cells or tissues. Anon-limiting example of a bioactive molecule is a protein. Otherexamples of the bioactive molecule include, but are not limited to,insulin, erythropoietin (EPO), glucagon-like peptide 1 (GLP-1), αMSH,parathyroid hormone (PTH), growth hormone, calcitonin, interleukin-2(IL-2), α1-antitrypsin, granulocyte/monocyte colony stimulating factor(GM-CSF), granulocyte colony stimulating factor (G-CSF), T20, anti-TNFantibodies, interferon α, interferon β, interferon γ, lutenizing hormone(LH), follicle-stimulating hormone (FSH), enkephalin, dalargin,kyotorphin, basic fibroblast growth factor (bFGF), hirudin, hirulog,lutenizing hormone releasing hormone (LHRH) analog, brain-derivednatriuretic peptide (BNP), or neurotrophic factors. The effector ofinterest can also be a glycosaminoglycan including, but not limited to,heparin, heparan sulfate, chondroitin sulfate, dermatan sulfate, andhyaluronic acid. The effector of interest can further be a nucleic acidsuch as DNA or RNA. Additionally, the effector can be a pharmaceuticallyactive agent, such as, for example, a toxin, a therapeutic agent, or anantipathogenic agent, such as an antibiotic, an antiviral, anantifungal, or an anti-parasitic agent. The effector of interest canitself be directly active or can be activated in situ by the peptide, bya distinct substance, or by environmental conditions.

The terms “pharmaceutically active agent” and “therapeutic agent” areused herein interchangeably to refer to a chemical material or compound,which, when administered to an organism, induces a detectablepharmacologic and/or physiologic effect.

The penetration compositions according to the present invention arecharacterized by the fact that their penetration capacity is virtuallyindependent of the nature of the effector that is coupled to it.

“Counter ions” according to this invention can include, for example,anionic or cationic amphipathic molecules, i.e., those having both polarand nonpolar domains, or both hydrophilic and hydrophobic properties.Anionic or cationic counter ions of this invention are ions that arenegatively (anionic) or positively (cationic) charged and can include ahydrophobic moiety. Under appropriate conditions, anionic or cationiccounter ions can establish electrostatic interactions with cationic oranionic impermeable molecules, respectively. The formation of such acomplex can cause charge neutralization, thereby creating a newuncharged entity, with further hydrophobic properties due to theinherent hydrophobicity of the counter ion.

Suitable anionic counter ions include ions with negatively chargedresidues derived from strong acids such as sulfonate or phosphonate, andfurther contain a hydrophobic moiety. Examples of such anionic counterions include, but are not limited to, sodium dodecyl sulphate anddioctyl sulfosuccinate.

Suitable cationic counter ions include quaternary amine derivatives,such as benzalkonium derivatives or other quaternary amines, which canbe substituted by hydrophobic residues. In general, quaternary aminescontemplated by the invention have the structure: 1-R1-2-R2-3-R3-4-R4-N,wherein R1, 2, 3, or 4 are alkyl or aryl derivatives. Further,quaternary amines can be ionic liquid forming cations, such asimidazolium derivatives, pyridinium derivatives, phosphonium compoundsor tetralkylammonium compounds.

For example, imidazolium derivatives have the general structure of1-R1-3-R2-imidazolium where R1 and R2 can be linear or branched alkylswith 1 to 12 carbons. Such imidazolium derivatives can be furthersubstituted for example by halogens or an alkyl group. Specificimidazolium derivatives include, but are not limited to,1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium,1-hexyl-3-methylimidazolium, 1-methyl-3-octylimidazolium,1-methyl-3-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoroctyl)-imidazolium,1,3-dimethylimidazolium, and 1,2-dimethyl-3-propylimidazolium.

Pyridinium derivatives have the general structure of1-R1-3-R2-pyridinium where R1 is a linear or branched alkyl with 1 to 12carbons, and R2 is H or a linear or branched alkyl with 1 to 12 carbons.Such pyridinium derivatives can be further substituted for example byhalogens or an alkyl group. Pyridinium derivatives include, but are notlimited to, 3-methyl-1-propylpyridinium, 1-butyl-3-methylpyridinium, and1-butyl-4-methylpyridinium.

In one embodiment, the present invention relates to the use of thecationic component of ionic liquids. Unlike other ionic liquids, thesalts of the cations according to the present invention are typicallywater soluble. For example, an anionic counterpart of the ionic liquidforming cation can be a halogen, such as chloride or bromide.

The penetration compositions of this invention involve the coupling ofthe penetrating peptide to the effector, directly or indirectly. As usedherein, the term “coupled” is meant to include all such specificinteractions that result in two or more molecules showing a preferencefor one another relative to some third molecule, including any type ofinteraction enabling a physical association between an effector and apenetrating peptide. Preferably this includes, but is not limited to,electrostatic interactions, hydrophobic interactions and hydrogenbonding, but does not include non-specific associations such as solventpreferences. The association must be sufficiently strong so that theeffector does not dissociate before or during penetration of thebiological barrier.

Furthermore, the coupling of the effector to the penetrating peptide canbe achieved indirectly via a mediator. For example, such a mediator canbe a large hydrophobic molecule, such as, for example, free fatty acids,mono-, di-, or tri-glycerides, ethers, or cholesterol esters of fattyacids, that binds the effector-counter ion complex, on the one hand, andthe hydrophobized penetrating peptide, on the other hand.

Also included in the invention are methods of producing penetrationcompositions. For example, a penetrating peptide or effector of thepenetration composition can be produced by standard recombinant DNAtechniques known in the art.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of vector is a “plasmid”, which refers to a circulardouble stranded DNA loop into which additional DNA segments can beligated. Another type of vector is a viral vector, wherein additionalDNA segments can be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) are integrated into the genome of a hostcell upon introduction into the host cell, and thereby are replicatedalong with the host genome. Moreover, certain vectors are capable ofdirecting the expression of genes to which they are operatively-linked.Such vectors are referred to herein as “expression vectors”. In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” can be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

Recombinant expression vectors comprise a nucleic acid in a formsuitable for expression of the nucleic acid in a host cell, which meansthat the recombinant expression vectors include one or more regulatorysequences, selected on the basis of the host cells to be used forexpression, that is operatively-linked to the nucleic acid sequence tobe expressed. Within a recombinant expression vector, “operably-linked”is intended to mean that the nucleotide sequence of interest is linkedto the regulatory sequence(s) in a manner that allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translationsystem or in a host cell when the vector is introduced into the hostcell).

The term “regulatory sequence” is intended to include promoters,enhancers and other expression control elements (e.g., polyadenylationsignals). Such regulatory sequences are described, for example, inGoeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, AcademicPress, San Diego, Calif. (1990). Regulatory sequences include those thatdirect constitutive expression of a nucleotide sequence in many types ofhost cell and those that direct expression of the nucleotide sequenceonly in certain host cells (e.g., tissue-specific regulatory sequences).It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice of thehost cell to be transformed, the level of expression of protein desired,etc. Expression vectors can be introduced into host cells to therebyproduce proteins or peptides encoded by nucleic acids as describedherein (e.g., penetrating peptides).

Recombinant expression vectors can be designed for expression ofpenetrating peptides or effectors of the invention in prokaryotic oreukaryotic cells. For example, penetrating peptides or effectors can beexpressed in bacterial cells such as Escherichia coli, insect cells(using baculovirus expression vectors), yeast cells or mammalian cells.Suitable host cells are discussed further in Goeddel, GENE EXPRESSIONTECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif.(1990). Alternatively, the recombinant expression vector can betranscribed and translated in vitro, for example using T7 promoterregulatory sequences and T7 polymerase.

Expression of proteins in prokaryotes is most often carried out inEscherichia coli with vectors containing constitutive or induciblepromoters directing the expression of either fusion or non-fusionproteins. Fusion vectors add a number of amino acids to a proteinencoded therein, usually to the amino terminus of the recombinantprotein. Such fusion vectors typically serve three purposes: (i) toincrease expression of recombinant protein; (ii) to increase thesolubility of the recombinant protein; and (iii) to aid in thepurification of the recombinant protein by acting as a ligand inaffinity purification.

Examples of suitable inducible non-fusion E. coli expression vectorsinclude pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d(Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185,Academic Press, San Diego, Calif. (1990) 60-89).

One strategy to maximize recombinant protein expression in E. coli is toexpress the protein in a host bacteria with an impaired capacity toproteolytically cleave the recombinant protein. See, e.g., Gottesman,GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press,San Diego, Calif. (1990) 119-128. Another strategy is to alter thenucleic acid sequence of the nucleic acid to be inserted into anexpression vector so that the individual codons for each amino acid arethose preferentially utilized in E. coli (see, e.g., Wada, et al., 1992.Nucl. Acids Res. 20: 2111-2118). Such alteration of nucleic acidsequences encoding the penetrating peptides or compositions of theinvention can be carried out by standard DNA synthesis techniques.

In another embodiment, the expression vector is a yeast expressionvector. Examples of vectors for expression in yeast Saccharomycescerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234),pMFa (Kurjan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz etal., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego,Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).

Alternatively, a penetrating peptide or effectors of the invention canbe expressed in insect cells using baculovirus expression vectors.Baculovirus vectors available for expression of proteins in culturedinsect cells (e.g., SF9 cells) include the pAc series (Smith, et al.,1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow andSummers, 1989. Virology 170: 31-39).

In yet another embodiment, a nucleic acid encoding the penetratingpeptides and effectors of the invention are expressed in mammalian cellsusing a mammalian expression vector. Examples of mammalian expressionvectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman,et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, theexpression vector's control functions are often provided by viralregulatory elements. For example, commonly used promoters are derivedfrom polyoma, adenovirus 2, cytomegalovirus, and simian virus 40. Forother suitable expression systems for both prokaryotic and eukaryoticcells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.

In another embodiment, the recombinant mammalian expression vector iscapable of directing expression of the nucleic acid preferentially in aparticular cell type (e.g., tissue-specific regulatory elements are usedto express the nucleic acid). Tissue-specific regulatory elements areknown in the art. Non-limiting examples of suitable tissue-specificpromoters include the albumin promoter (liver-specific; Pinkert, et al.,1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame andEaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of Tcell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) andimmunoglobulins (Banerji, et al., 1983. Cell 33: 729-740; Queen andBaltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., theneurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci.USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985.Science 230: 912-916), and mammary gland-specific promoters (e.g., milkwhey promoter; U.S. Pat. No. 4,873,316 and European ApplicationPublication No. 264,166). Developmentally-regulated promoters are alsoencompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990.Science 249: 374-379) and the α-fetoprotein promoter (Campes andTilghman, 1989. Genes Dev. 3: 537-546).

The invention further provides a recombinant expression vectorcomprising a DNA molecule encoding the penetrating peptides andeffectors of the invention cloned into the expression vector in anantisense orientation. That is, the DNA molecule is operatively-linkedto a regulatory sequence in a manner that allows for expression (bytranscription of the DNA molecule) of an RNA molecule that is antisenseto the penetrating peptide mRNA. Regulatory sequences operatively linkedto a nucleic acid cloned in the antisense orientation can be chosen thatdirect the continuous expression of the antisense RNA molecule in avariety of cell types, for instance viral promoters and/or enhancers, orregulatory sequences can be chosen that direct constitutive, tissuespecific or cell type specific expression of antisense RNA. Theantisense expression vector can be in the form of a recombinant plasmid,phagemid or attenuated virus in which antisense nucleic acids areproduced under the control of a high efficiency regulatory region, theactivity of which can be determined by the cell type into which thevector is introduced. For a discussion of the regulation of geneexpression using antisense genes see, e.g., Weintraub, et al.,“Antisense RNA as a molecular tool for genetic analysis,” Reviews-Trendsin Genetics, Vol. 1(1) 1986.

Another aspect of the invention pertains to host cells into which arecombinant expression vector has been introduced. The terms “host cell”and “recombinant host cell” are used interchangeably herein. It isunderstood that such terms refer not only to the particular subject cellbut also to the progeny or potential progeny of such a cell. Becausecertain modifications may occur in succeeding generations due to eithermutation or environmental influences, such progeny may not, in fact, beidentical to the parent cell, but are still included within the scope ofthe term as used herein.

A host cell can be any prokaryotic or eukaryotic cell. For example, thepenetrating peptide or effectors can be expressed in bacterial cellssuch as E. coli, insect cells, yeast or mammalian cells (such as Chinesehamster ovary cells (CHO) or COS cells). Other suitable host cells areknown to those skilled in the art.

Vector DNA can be introduced into prokaryotic or eukaryotic cells viaconventional transformation or transfection techniques. As used herein,the terms “transformation” and “transfection” are intended to refer to avariety of art-recognized techniques for introducing foreign nucleicacid (e.g., DNA) into a host cell, including calcium phosphate orcalcium chloride co-precipitation, DEAE-dextran-mediated transfection,lipofection, or electroporation. Suitable methods for transforming ortransfecting host cells can be found in Sambrook, et al. (MOLECULARCLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989),and other laboratory manuals.

For stable transfection of mammalian cells, it is known that, dependingupon the expression vector and transfection technique used, only a smallfraction of cells may integrate the foreign DNA into their genome. Inorder to identify and select these integrants, a gene that encodes aselectable marker (e.g., resistance to antibiotics) is generallyintroduced into the host cells along with the gene of interest. Variousselectable markers include those that confer resistance to drugs, suchas G418, hygromycin and methotrexate. Nucleic acid encoding a selectablemarker can be introduced into a host cell on the same vector as thatencoding the penetrating peptide or penetration composition, or can beintroduced on a separate vector. Cells stably transfected with theintroduced nucleic acid can be identified by drug selection (e.g., cellsthat have incorporated the selectable marker gene will survive, whilethe other cells die).

A host cell, such as a prokaryotic or eukaryotic host cell in culture,can be used to produce (i.e., express) a penetrating peptide or aneffector of the invention. Accordingly, the invention further providesmethods for producing penetrating peptides or effectors using the hostcells. In one embodiment, the method comprises culturing the host cell(into which a recombinant expression vector encoding a penetratingpeptide or an effector has been introduced) in a suitable medium suchthat the penetrating peptide or effector is produced. In anotherembodiment, the method further comprises isolating the penetratingpeptide or penetration composition from the medium or the host cell.

The penetrating peptides and effectors of the invention can also beproduced using solid-phase peptide synthesis methods known in the art.For example, a penetrating peptide can be synthesized using theMerrifield solid-phase synthesis method. (See e.g., Merrifield, R. B.,J. Am. Chem. Soc. 85:2149 (1963); ENCYCLOPEDIA OF MOLECULAR BIOLOGY 806(1st ed. 1994). In this method, the C-terminal amino acid is attached toan insoluble polymeric support resin (e.g., polystyrene beads), therebyforming an immobilized amino acid. To avoid unwanted reactions as theC-terminal amino acid is attached to the resin, the amino group of theC-terminal amino acid is protected or “blocked” using, for example, atert-butyloxylcarbonyl (t-BOC) group. The blocking group, e.g., t-BOC,on the immobilized amino acid is then removed by adding a dilute acid tothe solution. Before a second amino acid is attached to the immobilizedpeptide chain, the amino-group of the second amino acid is blocked, asdescribed above, and the α-carboxyl group of the second amino acid isactivated through a reaction with dicyclohxylcarbdiimide (DCC). Theactivated α-carboxyl group of the second amino acid then reacts with thefree amino group of the immobilized amino acid to form a peptide bond.Additional amino acids are then individually added to the terminal aminoacid of the immobilized peptide chain according to the required sequencefor the desired penetrating peptide or penetration composition. Once theamino acids have been added in the required sequence, the completedpeptide is released from the resin, such as for example, by usinghydrogen fluoride, which does not attack the peptide bonds.

The penetrating peptides or effectors of the invention can also besynthesized using Fmoc solid-phase peptide synthesis. (See e.g.,University of Illinois at Urbana-Champaign Protein Sciences Facility,Solid-Phase Peptide Synthesis (SPPS), athttp://www.biotech.uiuc.edu/spps.htm). In this method, the C-terminalamino acid is attached to an insoluble polymeric support resin (e.g.,polystyrene beads, cross-linked polystyrene resins, etc.), such as forexample, via an acid labile bond with a linker molecule. To avoidunwanted reactions as the C-terminal amino acid is being attached to theresin, the amino group of the C-terminal amino acid is blocked using anFmoc group. The blocking group, e.g., Fmoc, on the terminal amino acidof the immobilized amino acid is then removed by adding a base to thesolution. Side chain functional groups are also protected using anybase-stable, acid-labile groups to avoid unwanted reactions. Before thesecond amino acid is attached to the immobilized amino acid, theamino-group of the second amino acid is blocked, as described above, andthe α-carboxyl group of each successive amino acid is activated bycreating an N-hydrobenzotriazole (HOBt) ester in situ. The activatedα-carboxyl group of the second amino acid and the free amino group ofthe immobilized amino acid then react, in the presence of a base, toform a new peptide bond. Additional amino acids are then successivelyadded to the terminal amino acid of the immobilized peptide chain, untilthe desired peptide has been assembled. Once the necessary amino acidshave been attached, the peptide chain can be cleaved from the resin,such as for example, by using a mixture of trifluoroacetic acid (TFA)and scavengers (e.g., phenol, thioanisol, water, ethanedithiol (EDT) andtriisopropylsilan (TIS)) that are effective to neutralize any cationsformed as the protecting groups attached to the side chain functionalgroups of the assembled peptide chain are removed.

It is well known to those skilled in the art that proteins can befurther chemically modified to enhance the protein half-life incirculation. By way of non-limiting example, polyethylene glycol (PEG)residues can be attached to the penetrating peptides or effectors of theinvention. Conjugating biomolecules with PEG, a process known aspegylation, is an established method for increasing the circulatinghalf-life of proteins. Polyethylene glycols are nontoxic water-solublepolymers that, because of their large hydrodynamic volume, create ashield around the pegylated molecule, thereby protecting it from renalclearance, enzymatic degradation, as well as recognition by cells of theimmune system.

Agent-specific pegylation methods have been used in recent years toproduce pegylated molecules (e.g., drugs, proteins, agents, enzymes,etc.) that have biological activity that is the same as, or greaterthan, that of the “parent” molecule. These agents have distinct in vivopharmacokinetic and pharmacodynamic properties, as exemplified by theself-regulated clearance of pegfilgrastim, the prolonged absorptionhalf-life of pegylated interferon alpha-2a. Pegylated molecules havedosing schedules that are more convenient and more acceptable topatients, which can have a beneficial effect on the quality of life ofpatients. (See e.g., Yowell S. L. et al., Cancer Treat Rev 28 Suppl.A:3-6 (April 2002)).

The invention also includes methods of contacting biological barrierwith a penetration composition in an amount sufficient to enableefficient penetration of the compositions through the barrier. Thepenetration composition can be provided in vitro, ex vivo, or in vivo.Furthermore, the penetration composition according to this invention maybe capable of potentializing the biological activity of the coupledsubstance. Therefore, penetration compositions can be used to increasethe biological activity of the effector.

In addition to the penetration composition, the invention also providesa pharmaceutically acceptable base or acid addition salt, hydrate,ester, solvate, prodrug, metabolite, stereoisomer, or mixture thereof.The invention also includes pharmaceutical formulations comprising apenetration composition in association with a pharmaceuticallyacceptable carrier, diluent, protease inhibitor, surface active agent,or excipient. A surface active agent can include, for example,poloxamers, Solutol HS15, cremophore, or bile acids/salts.

Salts encompassed within the term “pharmaceutically acceptable salts”refer to non-toxic salts of the compounds of this invention which aregenerally prepared by reacting the free base with a suitable organic orinorganic acid or solvent to produce “pharmaceutically-acceptable acidaddition salts” of the compounds described herein. These compoundsretain the biological effectiveness and properties of the free bases.Representative of such salts are the water-soluble and water-insolublesalts, such as the acetate, amsonate(4,4-diaminostilbene-2,2′-disulfonate), benzenesulfonate, benzoate,bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calciumedetate, camsylate, carbonate, chloride, citrate, clavulariate,dihydrochloride, edetate, edisylate, estolate, esylate, fumarate,gluceptate, gluconate, glutamate, glycollylarsanilate,hexafluorophosphate, hexylresorcinate, hydrabamine, hydrobromide,hydrochloride, hydroxynaphthoate, iodide, isothionate, lactate,lactobionate, laurate, malate, maleate, mandelate, mesylate,methylbromide, methylnitrate, methylsulfate, mucate, napsylate, nitrate,N-methylglucamine ammonium salt, 3-hydroxy-2-naphthoate, oleate,oxalate, palmitate, pamoate (1,1-methylene-bis-2-hydroxy-3-naphthoate,embonate), pantothenate, phosphate/diphosphate, picrate,polygalacturonate, propionate, p-toluenesulfonate, salicylate, stearate,subacetate, succinate, sulfate, sulfosaliculate, suramate, tannate,tartrate, teoclate, tosylate, triethiodide, and valerate salts.

According to the methods of the invention, a patient, i.e., a humanpatient, can be treated with a pharmacologically or therapeuticallyeffective amount of a penetration composition. The term“pharmacologically or therapeutically effective amount” means thatamount of a drug or pharmaceutical agent (the effector) that will elicitthe biological or medical response of a tissue, system, animal or humanthat is being sought by a researcher or clinician.

The invention also includes pharmaceutical compositions suitable forintroducing an effector of interest across a biological barrier. Thecompositions are preferably suitable for internal use and include aneffective amount of a pharmacologically active compound of theinvention, alone or in combination, with one or more pharmaceuticallyacceptable carriers. The compounds are especially useful in that theyhave very low, if any, toxicity.

Preferred pharmaceutical compositions are tablets and gelatin capsules,enteric-coated, comprising the active ingredient together with a)diluents, e.g., lactose, dextrose, sucrose, mannitol, sorbitol,cellulose and/or glycine; b) protease inhibitors including, but notlimited to, aprotinin, Bowman-Birk inhibitor, soybean trypsin inhibitor,chicken ovomucoid, chicken ovoinhibitor, human pancreatic trypsininhibitor, camostate mesilate, flavonoid inhibitors, antipain,leupeptin, p-aminobenzamidine, AEBSF, TLCK, APMSF, DFP, PMSF,poly(acrylate) derivatives, chymostatin, benzyloxycarbonyl-Pro-Phe-CHO;FK-448, sugar biphenylboronic acids complexes, β-phenylpropionate,elastatinal, methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK)(SEQ ID NO:66), EDTA, chitosan-EDTA conjugates, amino acids,di-peptides, tripeptides, amastatin, bestatin, puromycin, bacitracin,phosphinic acid dipeptide analogues, α-aminoboronic acid derivatives,Na-glycocholate, 1,10-phenantroline, acivicin, L-serine-borate,thiorphan, and phosphoramidon; c) lubricants, e.g., silica, talcum,stearic acid, its magnesium or calcium salt, poloxamer and/orpolyethyleneglycol; for tablets also d) binders, e.g., magnesiumaluminum silicate, starch paste, gelatin, tragacanth, methylcellulose,sodium carboxymethylcellulose and/or polyvinylpyrrolidone; if desired e)disintegrants, e.g., starches, agar, alginic acid or its sodium salt, oreffervescent mixtures; and/or f) absorbents, colorants, flavors andsweeteners. The compositions may be sterilized and/or contain adjuvants,such as preserving, stabilizing, wetting or emulsifying agents, solutionpromoters, salts for regulating the osmotic pressure and/or buffers. Inaddition, they may also contain other therapeutically valuablesubstances. The compositions are prepared according to conventionalmixing, granulating or coating methods, respectively, and contain about0.01 to 75%, preferably about 0.1 to 10%, of the active ingredient.

Administration of the active compounds and salts described herein can bevia any of the accepted modes of administration for therapeutic agents.These methods include oral, bucal, anal, bronchial, nasal, transdermal,or topical administration modes. In general, those skilled in the artwill recognize that other, more invasive modes of administration, canalso be used. Such modes include, for example, parenteraladministration, i.e., subcutaneously, intraperitoneally, by viralinfection, intravascularly, intramuscularly, etc.

Depending on the intended mode of administration, the compositions maybe in solid, semi-solid or liquid dosage form, such as, for example,tablets, suppositories, pills, time-release capsules, powders, liquids,suspensions, aerosol or the like, preferably in unit dosages. Thecompositions will include an effective amount of active compound or thepharmaceutically acceptable salt thereof, and in addition, may alsoinclude any conventional pharmaceutical excipients and other medicinalor pharmaceutical drugs or agents, carriers, adjuvants, diluents,protease inhibitors, etc., as are customarily used in the pharmaceuticalsciences.

For solid compositions, excipients include pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum,cellulose, glucose, sucrose, magnesium carbonate, and the like may beused. The active compound defined above, may be also formulated assuppositories using for example, polyalkylene glycols, for example,propylene glycol, as the carrier.

Liquid compositions can, for example, be prepared by dissolving,dispersing, etc. The active compound is dissolved in or mixed with apharmaceutically pure solvent such as, for example, water, saline,aqueous dextrose, glycerol, ethanol, and the like, to thereby form thesolution or suspension.

If desired, the pharmaceutical composition to be administered may alsocontain minor amounts of non-toxic auxiliary substances such as wettingor emulsifying agents, pH buffering agents, and other substances such asfor example, sodium acetate, triethanolamine oleate, etc.

Those skilled in the art will recognize that the penetrationcompositions of the instant invention can also be used as an oral ornasal, i.e., mucosal, vaccine having an antigen, to which vaccination isdesired, serve as the effector. Such a vaccine may include a penetrationcomposition including a desired antigenic sequence, including, but notlimited to, the protective antigen (PA) component of Anthrax or theHepatitis B surface antigen (HBs) of Hepatitis B. This composition isthen orally or nasally administered to a subject in need of vaccination.

An “antigen” is a molecule or a portion of a molecule capable ofstimulating an immune response, which is additionally capable ofinducing an animal or human to produce antibody capable of binding to anepitope of that antigen. An “epitope” is that portion of any moleculecapable of being recognized by and bound by a major histocompatabilitycomplex (“MHC”) molecule and recognized by a T cell or bound by anantibody. A typical antigen can have one or more than one epitope. Thespecific recognition indicates that the antigen will react, in a highlyselective manner, with its corresponding MHC and T cell, or antibody andnot with the multitude of other antibodies which can be evoked by otherantigens.

A peptide is “immunologically reactive” with a T cell or antibody whenit binds to an MHC and is recognized by a T cell or binds to an antibodydue to recognition (or the precise fit) of a specific epitope containedwithin the peptide. Immunological reactivity can be determined bymeasuring T cell response in vitro or by antibody binding, moreparticularly by the kinetics of antibody binding, or by competition inbinding using known peptides containing an epitope against which theantibody or T cell response is directed as competitors.

Techniques used to determine whether a peptide is immunologicallyreactive with a T cell or with an antibody are known in the art.Peptides can be screened for efficacy by in vitro and in vivo assays.Such assays employ immunization of an animal, e.g., a mouse, a rabbit ora primate, with the peptide, and evaluation of the resulting antibodytiters.

Also included within the invention are vaccines that can elicit theproduction of secretory antibodies (IgA) against the correspondingantigen, as such antibodies serve as the first line of defense against avariety of pathogens. Oral or nasal i.e., mucosal, vaccination, whichhave the advantage of being non-invasive routes of administration, arethe preferred means of immunization for obtaining secretory antibodies,although those skilled in the art will recognize that the vaccinationcan be administered in a variety of ways, e.g., orally, topically, orparenterally, i.e., subcutaneously, intraperitoneally, by viralinfection, intravascularly, etc.

The compositions of the present invention can be administered in oraldosage forms such as tablets, capsules (each including timed release andsustained release formulations), pills, powders, granules, elixirs,tinctures, suspensions, syrups and emulsions.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counteror arrest the progress of the condition.

Oral dosages of the present invention, when used for the indicatedeffects, may be provided in the form of scored tablets containing 0.005,0.01, 0.025, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0,50.0, 100.0, 250.0, 500.0 or 1000.0 mg of active ingredient.

Compounds of the present invention may be administered in a single dailydose, or the total daily dosage may be administered in divided doses oftwo, three or four times daily. Furthermore, preferred compounds for thepresent invention can be administered in bucal form via topical use ofsuitable bucal vehicles, bronchial form via suitable aerosols orinhalants, intranasal form via topical use of suitable intranasalvehicles, or via transdermal routes, using those forms of transdermalskin patches well known to those of ordinary skill in that art. To beadministered in the form of a transdermal delivery system, the dosageadministration will, of course, be continuous rather than intermittentthroughout the dosage regimen. Other preferred topical preparationsinclude creams, ointments, lotions, aerosol sprays and gels, wherein theconcentration of active ingredient would range from 0.1% to 15%, w/w orw/v.

The compounds herein described in detail can form the active ingredient,and are typically administered in admixture with suitable pharmaceuticaldiluents, excipients or carriers (collectively referred to herein as“carrier” materials) suitably selected with respect to the intended formof administration, that is, oral tablets, capsules, elixirs, syrups andthe like, and consistent with conventional pharmaceutical practices.

For instance, for oral administration in the form of a tablet orcapsule, the active drug component can be combined with an oral,non-toxic pharmaceutically acceptable inert carrier such as ethanol,glycerol, water and the like. Moreover, when desired or necessary,suitable binders, lubricants, protease inhibitors, disintegrating agentsand coloring agents can also be incorporated into the mixture. Suitablebinders include starch, gelatin, natural sugars such as glucose orbeta-lactose, corn sweeteners, natural and synthetic gums such asacacia, tragacanth or sodium alginate, carboxymethylcellulose,poloxamer, polyethylene glycol, waxes and the like. Lubricants used inthese dosage forms include sodium oleate, sodium stearate, magnesiumstearate, sodium benzoate, sodium acetate, sodium chloride and the like.Disintegrators include, without limitation, starch, methylcellulose,agar, bentonite, xanthan gum and the like.

The compounds of the present invention may also be coupled with solublepolymers as targetable drug carriers. Such polymers can includepolyvinylpyrrolidone, pyran copolymer,polyhydroxypropyl-methacrylamide-phenol,polyhydroxyethylaspanamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds of thepresent invention may be coupled to a class of biodegradable polymersuseful in achieving controlled release of a drug, for example,polylactic acid, polyepsilon caprolactone, polyhydroxy butyric acid,polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates andcross-linked or amphipathic block copolymers of hydrogels.

Any of the above pharmaceutical compositions may contain 0.01-99%,preferably 0.1-10% of the active compounds as active ingredients.

The following EXAMPLES are presented in order to more fully illustratethe preferred embodiments of the invention. These EXAMPLES should in noway be construed as limiting the scope of the invention, as defined bythe appended claims.

EXAMPLES Example 1 Utilization of the Penetration Composition to Enablethe Translocation of Aminoglycoside Antibiotics Across an EpithelialBarrier

SEQ ID NO: 34 (or any other sequence from SEQ ID NO:22, 30-37) ishydrophobized via acylation of the free amino groups of the two lysineresidues at the C-terminus of the penetrating peptide with a fatty acid,i.e., myristoyl. Similarly, any other sequence from SEQ ID NO: 1-15,24-29 may be also supplemented by extra lysine residues, interspaced byglycine, alanine or serine residues, added at the penetrating peptideC-terminus, and the free amino groups of such lysine residues areacylated with a fatty acid. The hydrophobized peptide is thenincorporated into the penetrating composition, which further contains alyophilizate of (1) an aminoglycoside antibiotic, i.e., gentamycin, (2)an amphipathic counter anion, such as sodium dodecyl sulfate (SDS) ordioctyl sulfosuccinate (DSS) and (3) phytic acid. Additionalconstituents are specified in Table 3. TABLE 3 Additional constituentsof the penetration composition N-Methyl Pirolidone (NMP) Cremophor ELTricaprine Pluronic F-68 Aprotinin Solutol HS-15 (SHS) N-Acetyl Cysteine(NAC)

The penetration composition is then administered to test animals, i.e.mice, in two forms: rectally or by injection into an intestinal loop.The experimental procedure involves male BALB/c mice, which are deprivedof food, 18 hours prior to the experiment. For intra-intestinalinjection the mice are then anesthetized and a 2 cm long incision ismade along the center of the abdomen, through the skin and abdominalwall. An intestine loop is gently pulled out through the incision andplaced on wet gauze beside the animal. The loop remains intact throughthe entire procedure and is kept wet during the whole time. The testedcompound is injected into the loop, using a 26 G needle. For rectaladministration the the mice are anesthetized and the penetrationcomposition is then rectally administered to the mice, 100 μl/mouse,using a plastic tip covered with a lubricant.

Penetration is assessed in two methods: (a) direct measurement ofantibiotic concentrations in the blood, and (b) measurement ofantibacterial activity in serum samples from treated animals.

Example 2 Utilization of the Penetration Composition to Enable theTranslocation of Cationic Antifungal Agents Such as Caspofungin Acrossan Epithelial Barrier

SEQ ID NO: 34 (or any other sequence from SEQ ID NO:22, 30-37) ishydrophobized via acylation of the free amino groups of the two lysineresidues at the C-terminus of the penetrating peptide with a fatty acid,i.e., myristoyl. Similarly, any other sequence from SEQ ID NO: 1-15,24-29 may be also supplemented by extra lysine residues, interspaced byglycine, alanine or serine residues, added at the penetrating peptideC-terminus, and the free amino groups of such lysine residues areacylated with a fatty acid. The hydrophobized peptide is thenincorporated into the penetrating composition, which further contains alyophilizate of (1) an antifungal agent, i.e., caspofungin, (2) anamphipathic counter anion, such as sodium dodecyl sulfate (SDS) ordioctyl sulfosuccinate (DSS) and (3) phytic acid. Additionalconstituents are specified in Table 4. TABLE 4 Additional constituentsof the penetration composition N-Methyl Pirolidone (NMP) Cremophor ELTricaprine Pluronic F-68 Aprotinin Solutol HS-15 (SHS) N-Acetyl Cysteine(NAC)

The penetration composition is then administered to test animals, i.e.,mice, in two forms: rectally or by injection into an intestinal loop.The experimental procedure involves male BALB/c mice, which are deprivedof food, 18 hours prior to the experiment. For intra-intestinalinjection the mice are then anesthetized and a 2 cm long incision ismade along the center of the abdomen, through the skin and abdominalwall. An intestine loop is gently pulled out through the incision andplaced on wet gauze beside the animal. The loop remains intact throughthe entire procedure and is kept wet during the whole time. The testedcompound is injected into the loop, using a 26 G needle. For rectaladministration the mice are anesthetized and the penetration compositionis then rectally administered, 100 μl/mouse, using a plastic tip coveredwith a lubricant.

Penetration is assessed in two methods: (a) direct measurement ofcaspofungin concentrations in the blood, and (b) measurement ofantifungal activity in serum samples from treated animals.

Example 3 Utilization of the Penetration Composition for MucosalVaccination

SEQ ID NO: 34 (or any other sequence from SEQ ID NO:22, 30-37) ishydrophobized via acylation of the free amino groups of the two lysineresidues at the C-terminus of the penetrating peptide with a fatty acid,i.e., myristoyl. Similarly, any other sequence from SEQ ID NO: 1-15,24-29 may be also be supplemented by extra lysine residues, interspacedby glycine, alanine or serine residues, added at the penetrating peptideC-terminus, and the free amino groups of such lysine residues areacylated with a fatty acid. The hydrophobized peptide is thenincorporated into the penetrating composition, which further contains alyophilizate of (1) a desired antigenic sequence, e.g., the HBs antigenof Hepatitis B, (2) an amphipathic counter anion, such as sodium dodecylsulfate (SDS) or dioctyl sulfosuccinate (DSS) and (3) phytic acid.Additional constituents are specified in Table 3. Such a pharmaceuticalcomposition can be administered to a subject in need of vaccination.

This method allows simple and rapid vaccination of large populations inneed thereof. Another advantage of this method is the production of hightiters of IgA antibodies and the subsequent presence of IgA antibodiesin the epithelial mucosa, which are the sites of exposure to antigens.

Efficacy of vaccination can be demonstrated by the measurement ofspecific antibody titers, IgA in particular, as well as the measurementof immunological response to stimulation, such as for example, via acutaneous hypersensitivity reaction in response to subcutaneousadministration of antigen.

Example 4 Utilization of the Penetration Composition to Enable theTranslocation of Heparin Across an Epithelial Barrier

SEQ ID NO: 36 was hydrophobized via acylation of the free amino groupsof the two lysine residues at the C-terminus of the penetrating peptidewith a myristoyl. Acylation with myristoyl was achieved by incubatingthe peptide with myristoyl chloride in a molar ratio of 1:10, underbasic pH conditions in the presence of appropriate solvents (benzylbenzoate and di-methyl formamide, with 1% bicarbonate). Thehydrophobized peptide was then incorporated into the penetrationcomposition, which further contained heparin, and the counter cation1-butyl-3-methylimidazolium chloride (BMIC). Additional components ofthe penetration composition are specified in Table 5. TABLE 5Penetration composition for heparin translocation Hydrophobized SEQ IDNO: 36 7.5 μl/ml Heparin 10 mg/ml 1-butyl-3-methylimidazolium chloride4% (BMIC) N-Methyl Pirolidone (NMP) 10%  Cremophor EL 0.37%   Tricaprine0.5%   Pluronic F-68 2% Aprotinin 20 μl/ml Solutol HS-15 (SHS) 2%N-Acetyl Cysteine (NAC) 5 μgr/mlIn Vivo Experimental Procedure:

Four male BALB/c mice, 9-10 weeks old, were deprived of food, 18 hoursprior to the experiment. The mice were anesthetized by i.p. injection of0.05 ml of a mixture of 0.15 ml xylazine+0.85 ml of ketamin. Thepenetration composition was then rectally administered to the mice, 100μl/mouse, using a plastic tip covered with a lubricant. Penetration wasassessed via measurement of clotting time, at different time intervalsafter heparin administration. Five minutes post administration the tipof the tail was cut and a 50 μl blood sample was drawn into a glasscapillary. The capillary was broken at different time intervals, untilclot formation was observed. This was repeated at 15, 30, 60, 90, 120and 150 minutes post administration. The animals were subsequentlysacrificed.

In similar experiments, a control peptide (SEQ ID NO:16), lacking thepenetrating peptide-sequence, was similarly hydrophobized andincorporated into the penetration composition of Table 5 and thenrectally administered to the mice. The average clotting time measuredwas only slightly elongated compared to that obtained with the fullconjugate of the penetrating peptide. Results are shown in Table 6.TABLE 6 Sample Clotting time, measured at follow times after injectionMouse # injected 0 5 min 15 min 30 min 60 min 90 min 120 min 150 min 1SEQ ID 1′ 1′ 1′ 2′ 5′ 4′  2′  3′ NO: 16 2 SEQ ID 1.5′ 1′ 1′ 1.5′ 2.5′ 5′ 3′  4′ NO: 36 3 SEQ ID 2.5′ 2′ 1′ 3′ 6′ 9′*  8′*  6′ NO: 36 4 SEQ ID1.5′ 1′ 1.5′ 1.5′ 8′* 9′* 15′*  17′*  NO: 36 5 SEQ ID 1′ 2′ 3′ 2′ 9′*7′*  7′*  9′* NO: 36*indicates appearance of blood clotting, but it did not progress evenafter several minutes.

Clotting time values increase in relation to the amount of heparinabsorbed from the intestine into the bloodstream (i.e., in an amountthat correlates to the amount of heparin absorbed). Therefore, this drugdelivery system will replace the use of heparin injections.

Example 5 Utilization of the Penetration Composition to Enable theTranslocation of Insulin Across an Epithelial Barrier using HMIC as theCounter Ion

SEQ ID NO: 36 (also called IBW-002V2) and SEQ ID NO: 16 (also calledIBW-001) were hydrophobized via acylation of the free amino groups ofthe two lysine residues at the C-terminus of the penetrating peptideswith a myristoyl. Acylation with myristoyl was achieved by incubatingthe peptide with myristoyl chloride in a molar ratio of 1:10, underbasic pH conditions in the presence of appropriate solvents (benzylbenzoate and di-methyl formamide, with 1% bicarbonate). Thehydrophobized peptides were then incorporated into the penetrationcomposition, which further contained insulin, and the counter cation1-hexyl-3-methylimidazolium chloride (HMIC). Additional components ofthe penetration composition are specified in Table 7. TABLE 7Penetration composition for insulin translocation Hydrophobized PeptideInsulin 1-hexyl-3-methylimidazolium chloride (HMIC) NaOH Acetic acidSodium Acetate L-arginine Pluronic F-68 Aprotinin Solutol HS-15 (SHS)N-Acetyl Cysteine (NAC)

Eight male BALB/c mice, 9-10 weeks old, were deprived of food, 18 hoursprior to the experiment. The animals were divided into 4 groups. Eachpreparation was administered to 2 groups of mice either i.p. (70ul/mouse, containing 0.2 IU insulin) or rectal (70 ul/mouse, containing0.2 IU insulin). Blood glucose levels were measured at various timeintervals post administration, in blood samples drawn from the tip ofthe tail. Glucose levels were plotted against time post insulinadministration (See FIG. 3).

As can be seen in FIG. 3, after the penetrating peptide composition withIBW-002V2 was administered, glucose levels dropped gradually andsignificantly, in both groups, indicating insulin absorption from theintestine into the blood stream. In contrast, with the control peptidecomposition (IBW-001) a significant drop in glucose levels was noticedonly after i.p. administration. No change in blood glucose levels wasobserved after rectal administration, indicating there was no insulinabsorption in this group.

Blood glucose levels decrease in relation to the amount of insulinabsorbed from the intestine into the bloodstream (i.e., in an amountthat correlates to the amount of insulin absorbed). Thus, this drugdelivery system can replace the need for insulin injections, therebyproviding an efficient, safe and convenient route of administration fordiabetes patients.

Example 6 Utilization of the Penetration Composition for MucosalVaccination

SEQ ID NO: 34 (or any other sequence from SEQ ID NO:22, 30-37) ishydrophobized via acylation of the free amino groups of the two lysineresidues at the C-terminus of the penetrating peptide with a fatty acid,i.e., myristoyl. Similarly, any other sequence from SEQ ID NO: 1-15,24-29 may also be supplemented by extra lysine residues, interspaced byglycine, alanine or serine residues, added at the penetrating peptideC-terminus, and the free amino groups of such lysine residues areacylated with a fatty acid. The hydrophobized peptide is thenincorporated into the penetrating composition, which further contains alyophilizate of (1) a desired antigenic sequence, e.g., the PA antigenof Anthrax, (2) an amphipathic counter cation, such as1-butyl-3-methylimidazolium chloride (BMIC) or1-hexyl-3-methylimidazolium chloride (HMIC) and (3) phytic acid.Additional constituents are specified in Table 5. Such a pharmaceuticalcomposition can be administered to a subject in need of vaccination.

This method allows simple and rapid vaccination of large populations inneed thereof. Another advantage of this method is the production of hightiters of IgA antibodies and the subsequent presence of IgA antibodiesin the epithelial mucosa, which are the sites of exposure to antigens.

Efficacy of vaccination can be demonstrated by the measurement ofspecific antibody titers, IgA in particular, as well as the measurementof immunological response to stimulation, such as for example, via acutaneous hypersensitivity reaction in response to subcutaneousadministration of antigen.

Example 7 Utilization of the Penetration Composition to Enable theTranslocation of Insulin Across an Epithelial Barrier Using BKC as theCounter Ion

SEQ ID NO: 36 (also called IBW-002V2) was hydrophobized via acylation ofthe free amino groups of the two lysine residues at the C-terminus ofthe penetrating peptide with a myristoyl. Acylation with myristoyl wasachieved by incubating the peptide with myristoyl chloride in a molarratio of 1:10, under basic pH conditions in the presence of appropriatesolvents (benzyl benzoate and di-methyl formamide, with 1% bicarbonate).The hydrophobized peptide was then incorporated into the penetrationcomposition, which further contained a lyophilizate of (1) insulin, (2)the counter cation Benzalkonium Chloride (BKC), and (3) phytic acid at aratio of 1:0.5:0.5. Additional components of the penetration compositionare specified in Table 8. TABLE 8 Penetration composition for insulintranslocation Hydrophobized Peptide Human Insulin Benzalkonium Chloride(BKC) Phytic Acid NaOH Acetic acid Sodium Acetate L-arginine PluronicF-68 Aprotinin Solutol HS-15 (SHS) N-Acetyl Cysteine (NAC) TricaprineEthanol

Twelve male SD rats, 160-190 gr, were deprived of food, 18 hours priorto the experiment. The animals were divided into groups. Thepreparations were administered as follows: Rats #1,2—rectal PBS 200 ul,rats #3,4—rectal 200 ul penetration composition as specified abovewithout peptide (5 IU insulin), rat #5—i.p. 200 ul penetrationcomposition with peptide (1 IU insulin), rats #6,7—rectal 200 ulpenetration composition with peptide (5 IU insulin). Blood glucoselevels were measured at various time intervals post administration, inblood samples drawn from the tip of the tail. Glucose levels wereplotted against time post insulin administration (See FIG. 4). TABLE 9glucose (mg/dL), time post administration 0 15 30 45 60 90 rat # 1 79 9885 80 74 70 rat # 2 58 93 91 80 72 69 rat # 3 83 67 80 77 72 72 rat # 4106 110 107 99 105 93 rat # 5 80 77 50 33 10 10 rat # 6 85 79 55 35 2133 rat # 7 93 78 53 39 23 3110 = low

As can be seen in FIG. 4, after the penetrating peptide composition withIBW-002V2 was rectally administered, glucose levels dropped graduallyand significantly, in both rats, indicating insulin absorption from theintestine into the blood stream. In contrast, without the peptide asignificant drop in glucose levels was noticed only after i.p.administration. No change in blood glucose levels was observed afterrectal administration, indicating there was no insulin absorption inthese rats.

Blood glucose levels decrease in relation to the amount of insulinabsorbed from the intestine into the bloodstream (i.e., in an amountthat correlates to the amount of insulin absorbed). Thus, this drugdelivery system can replace the need for insulin injections, therebyproviding an efficient, safe and convenient route of administration fordiabetes patients.

OTHER EMBODIMENTS

From the foregoing detailed description of the specific embodiments ofthe invention, it should be apparent that unique methods oftranslocation across epithelial and endothelial barriers have beendescribed. Although particular embodiments have been disclosed herein indetail, this has been done by way of example for purposes ofillustration only, and is not intended to be limiting with respect tothe scope of the appended claims that follow. In particular, it iscontemplated by the inventor that various substitutions, alterations,and modifications may be made to the invention without departing fromthe spirit and scope of the invention as defined by the claims. Forinstance, the choice of the particular type of tissue, or the particulareffector to be translocated is believed to be a matter of routine for aperson of ordinary skill in the art with knowledge of the embodimentsdescribed herein.

1. A composition for non-invasive translocation of at least one effectoracross a biological barrier, said composition comprising: (a) atherapeutically effective amount of said at least one effector; (b) acounter ion to the at least one effector; and (c) an Escherichia colirelated penetrating peptide wherein said penetrating peptide ishydrophobized.
 2. The composition of claim 1, wherein the penetratingpeptide comprises an amino acid sequence selected from the groupconsisting of: SEQ ID NOS: 3, 25, 26, and
 27. 3. The composition ofclaim 1 further comprising a pharmaceutically acceptable carrier,diluent, or excipient, or a combination thereof.
 4. The composition ofclaim 3, wherein said composition is in the form of a capsule or tablet.5. The composition of claim 4, wherein said capsule or tablet isenteric-coated.
 6. The composition of claim 3, wherein said compositionis in the form selected from the group consisting of an aqueousdispersion, a suspension, and an emulsion.
 7. The composition of claim3, wherein said composition is in the form of a cream.
 8. Thecomposition of claim 3, wherein said composition is in the form of anointment.
 9. The composition of claim 3, wherein said composition is inthe form of a suppository.
 10. The composition of claim 1, wherein saidat least one effector is an impermeable molecule.
 11. The composition ofclaim 10, wherein said impermeable molecule is cationic.
 12. Thecomposition of claim 10, wherein said impermeable molecule is anionic.13. The composition of claim 12, wherein said anionic impermeablemolecule is a polysaccharide.
 14. The composition of claim 13, whereinsaid polysaccharide is a glycosaminoglycan.
 15. The composition of claim14, wherein said glycosaminoglycan is selected from the group consistingof: heparin; heparan sulfate; chondroitin sulfate; dermatan sulfate;hyaluronic acid and pharmaceutically acceptable salts thereof.
 16. Thecomposition of claim 1, wherein said at least one effector is apharmaceutically active agent selected from the group consisting of: ahormone, a growth factor, a neurotrophic factor, an anticoagulant, abioactive molecule, a toxin, an antibiotic, an anti-fungal agent, anantipathogenic agent, an antigen, an antibody, an antibody fragment, animmunomodulator, a vitamin, an antineoplastic agent, an enzyme, and atherapeutic agent.
 17. The composition of claim 16, wherein saidbioactive molecule is selected from the group consisting of: insulin;erythropoietin (EPO); glucagon-like peptide 1 (GLP-1); αMSH; parathyroidhormone (PTH); growth hormone; calcitonin; interleukin-2 (IL-2);α1-antitrypsin; granulocyte/monocyte colony stimulating factor (GM-CSF);granulocyte colony stimulating factor (G-CSF); T20; anti-TNF antibodies;interferon α; interferon β; interferon γ; lutenizing hormone (LH);follicle-stimulating hormone (FSH); enkephalin; dalargin; kyotorphin;basic fibroblast growth factor (bFGF); hirudin; hirulog; lutenizinghormone releasing hormone (LHRH) analog; brain-derived natriureticpeptide (BNP); and neurotrophic factors.
 18. The composition of claim16, wherein said anti-fungal agent is Caspofungin.
 19. The compositionof claim 16, wherein said vitamin is vitamin B12.
 20. The composition ofclaim 16, wherein said antibiotic is an aminoglycoside antibiotic. 21.The composition of claim 20, wherein said aminoglycoside antibiotic isselected from the group consisting of Gentamycin, Amikacin, Tobramycin,and Neomycin.
 22. The composition of claim 1, wherein said effector is anucleic acid or a nucleic acid mimetic.
 23. The composition of claim 1,wherein said counter ion is an amphipathic molecule.
 24. The compositionof claim 23, wherein said amphipathic molecule is cationic.
 25. Thecomposition of claim 23, wherein said amphipathic molecule is anionic.26. The composition of claim 25, wherein said anionic amphipathicmolecule is derived from a strong acid selected from the groupconsisting of sulfonate and phosphonate, and wherein said anionicamphipathic molecule further comprises a hydrophobic moiety.
 27. Thecomposition of claim 25, wherein the anionic amphipathic molecule isselected from the group consisting of: sodium dodecyl sulphate anddioctyl sulfosuccinate.
 28. The composition of claim 24, wherein saidcationic amphipathic molecule is a quaternary amine comprising ahydrophobic moiety.
 29. The composition of claim 28, wherein saidquaternary amine has the general structure of:

wherein R1, R2, R3 and R4 are alkyl or aryl residues.
 30. Thecomposition of claim 29, wherein said quaternary amine is a benzalkoniumderivative.
 31. The composition of claim 1, wherein said counter ion isan ionic liquid forming cation.
 32. The composition of claim 31, whereinsaid ionic liquid forming cation is selected from the group consistingof: imidazolium derivatives; pyridinium derivatives; phosphoniumcompounds; and tetralkylammonium compounds.
 33. The composition of claim32, wherein said imidazolium derivative has the general structure of1-R1-3-R2-imidazolium, wherein R1 and R2 are linear or branched alkylswith 1 to 12 carbons.
 34. The composition of claim 33, wherein saidimidazolium derivatives further comprise a halogen or an alkyl groupsubstitution.
 35. The composition of claim 33, wherein said imidazoliumderivative is selected from the group consisting of:1-ethyl-3-methylimidazolium; 1-butyl-3 methylimidazolium;1-hexyl-3-methylimidazolium; 1-methyl-3-octylimidazolium;1-methyl-3-(3,3,4,4,5,5,6,6,7,7,8,8,8-tridecafluoroctyl)-imidazolium;1,3-dimethylimidazolium; and 1,2-dimethyl-3-propylimidazolium.
 36. Thecomposition of claim 32, wherein said pyridinium derivative has thegeneral structure of 1-R1-3-R2-pyridinium, wherein R1 is a linear orbranched alkyl with 1 to 12 carbons, and R2 is H or a linear or branchedalkyl with 1 to 12 carbons.
 37. The composition of claim 36, whereinsaid pyridinium derivatives further comprise a halogen or an alkyl groupsubstitution.
 38. The composition of claim 36, wherein said pyridiniumderivative is selected from the group consisting of3-methyl-1-propylpyridinium, 1-butyl-3-methylpyridinium, and1-butyl-4-methylpyridinium.
 39. The composition of claim 1, furthercomprising a polyanionic molecule.
 40. The composition of claim 39,wherein said polyanionic molecule is phytic acid.
 41. The composition ofclaim 1, further comprising a surface active agent.
 42. The compositionof claim 41, wherein said surface active agent is selected from thegroup consisting of a poloxamer, Solutol HS15, Cremophore, and bileacids.
 43. The composition of claim 1, wherein said composition isdissolved in an at least partially water soluble solvent.
 44. Thecomposition of claim 43, wherein said at least partially water solublesolvent is selected from the group consisting of: n-butanol; isoamyl(=isopentyl) alchohol; iso-butanol; iso-propanol; propanol; ethanol;ter-butanol alcohols; polyols; DMF; DMSO; ethers; amides; esters; andmixtures thereof.
 45. The composition of claim 1, wherein any one ormore of the components of the composition is lyophilized.
 46. Thecomposition of claim 1, wherein said composition further comprises ahydrophobic carrier comprising at least one hydrophobic molecules,wherein said molecules are aliphatic, aromatic, or mixtures thereof. 47.The composition of claim 46, wherein said aliphatic hydrophobicmolecules are selected from the group consisting of fatty acids,monoglycerides, diglycerides, triglycerides, ethers, and cholesterolesters of fatty acids.
 48. The composition of claim 47, wherein saidtriglyceride is tricaprin.
 49. The composition of claim 46, wherein saidaromatic hydrophobic molecule is benzyl benzoate.
 50. The composition ofclaim 1, further comprising at least one protective agent.
 51. Thecomposition of claim 50, wherein said protective agent is a proteaseinhibitor selected from the group consisting of: aprotinin; Bowman-Birkinhibitor; soybean trypsin inhibitor; chicken ovomucoid; chickenovoinhibitor; human pancreatic trypsin inhibitor; camostate mesilate;flavonoid inhibitors; antipain; leupeptin; paminobenzamidine; AEBSF;TLCK; APMSF; DFP; PMSF; poly(acrylate) derivatives; chymostatin;benzyloxycarbonyl-Pro-Phe-CHO; FK-448; sugar biphenylboronic acidscomplexes; β-phenylpropionate; elastatinal;methoxysuccinyl-Ala-Ala-Pro-Valchloromethylketone (MPCMK); EDTA;chitosan-EDTA conjugates; amino acids; dipeptides; tripeptides;amastatin; bestatin; puromycin; bacitracin; phosphinic acid dipeptideanalogues; α-aminoboronic acid derivatives; Na-glycocholate;1,10-phenantroline; acivicin; L-serine-borate; thiorphan; andphosphoramidon.
 52. The composition of claim 1, wherein the penetratingpeptide is further modified, via one or more peptidic bonds, to enableprotection from gastrointestinal proteolysis.
 53. The composition ofclaim 1, wherein said penetrating peptide further contains lysineresidues, interspaced by glycine, alanine or serine residues, added atthe C-terminus of the penetrating peptide, and wherein the free aminogroups of said lysine residues are acylated.
 54. The composition ofclaim 53, wherein acylation utilizes long-chain fatty acids selectedfrom the group of: stearoyl, palmitoyl, oleyl, ricinoleyl, lauroyl andmyristoyl.
 55. The composition of claim 3, wherein the compositionfurther comprises two or more of the following: a non-ionic detergent,an ionic detergent, a protease inhibitor, and a reducing agent.
 56. Thecomposition of claim 55, wherein the non-ionic detergent is a poloxameror polyehtyleneglycol-15-hydroxystearate (Solutol HS15).
 57. Thecomposition of claim 56, wherein the poloxamer ispolyoxyethylene-polyoxypropylene block copolymer.
 58. The composition ofclaim 55, wherein the ionic detergent is a bile salt.
 59. Thecomposition of claim 58, wherein the bile salt is Taurodeoxycholate. 60.The composition of claim 55, wherein the protease inhibitor is selectedfrom the group consisting of: aprotinin; Bowman-Birk inhibitor; soybeantrypsin inhibitor; chicken ovomucoid; chicken ovoinhibitor; humanpancreatic trypsin inhibitor; camostate mesilate; flavonoid inhibitors;antipain; leupeptin; p-aminobenzamidine; AEBSF; TLCK; APMSF; DFP; PMSF;poly(acrylate) derivatives; chymostatin; benzyloxycarbonyl-Pro-Phe-CHO;FK-448; sugar biphenylboronic acids complexes; β-phenylpropionate;elastatinal; methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone (MPCMK);EDTA; chitosan-EDTA conjugates; amino acids; dipeptides; tripeptides;amastatin; bestatin; puromycin; bacitracin; phosphinic acid dipeptideanalogues; α-aminoboronic acid derivatives; Na-glycocholate;1,10-phenantroline; acivicin; L-serine-borate; thiorphan; andphosphoramidon.
 61. The composition of claim 55, wherein the reducingagent is N-acetyl-L-cystein (NAC).
 62. A kit for treating or preventinga disease or pathological condition comprising, in one or morecontainers, a therapeutically or prophylactically effective amount ofthe composition of claim
 3. 63. The composition of claim 1, wherein saidpenetrating peptide further comprises a chemical modification.
 64. Thecomposition of claim 63, wherein the chemical modification comprises theattachment of one or more polyethylene glycol residues to thepenetrating peptide.
 65. A method of mucosal vaccination, the methodcomprising administering to a subject in need of vaccination thecomposition of claim 3, wherein the at least one effector comprises anantigen to which vaccination is desired.
 66. The method of claim 65,wherein the antigen to which vaccination is desired is selected from thegroup consisting of PA for use in a vaccine against Anthrax, and HBs foruse in a vaccine against Hepatitis B.
 67. A method of treating orpreventing a disease or pathological condition, said method comprisingadministering to a subject in which such treatment or prevention isdesired, the composition of claim 3, in an amount sufficient to treat orprevent said disease or said pathological condition in said subject. 68.The method of claim 67, wherein said disease or said pathologicalcondition is selected from the group consisting of: endocrine disorders;diabetes; infertility; hormone deficiencies; osteoporosis;neurodegenerative disorders; Alzheimer's disease; dementia; Parkinson'sdisease; multiple sclerosis; Huntington's disease; cardiovasculardisorders; atherosclerosis; hyper- and hypocoagulable states; coronarydisease; cerebrovascular events; metabolic disorders; obesity; vitamindeficiencies; renal disorders; renal failure; haematological disorders;anemia of different entities; immunologic and rheumatologic disorders;autoimmune diseases; immune deficiencies; infectious diseases; viralinfections; bacterial infections; fungal infections; parasiticinfections; neoplastic diseases; multi-factorial disorders; impotence;chronic pain; depression; different fibrosis states; and short stature.